Figure 2
SHP suppresses BMP6-induced hepcidin gene expression.
(a) Q-PCR analysis showing hepcidin and SHP mRNA levels in AML12 cells treated with BMP6 (20 nM). (b) Q-PCR analysis showing hepcidin and SHP mRNA levels in mouse hepatocytes treated with BMP6 (20 nM) for 24 h. (c) Western blot analysis, showing SHP and AMPK expression in HepG2 cells infected with Ad-GFP, Ad-Flag-SHP and Ad-AMPK ca for 24 h +, 10 MOI. (d) Q-PCR analysis showing hepcidin mRNA levels in hepatocytes isolated from wild-type and SHP knockout mice. Hepatocytes were treated with BMP6 (20 nM) and infected with Ad-GFP, Ad-AMPK ca or Ad-Flag-SHP for 24 h. (e) Inhibitory effect of SHP on BMP6-mediated induction of mouse and human hepcidin promoter activity. HepG2 cells were transfected with vectors expressing mouse Hepcidin-luc and mSHP or human Hepcidin-luc and hSHP, and treated with BMP6 (20 nM) for 24 h. 2, 200 ng; 4, 400 ng; 6, 600 ng. (f) Q-PCR analysis showing hepcidin (left panel) and SHP (right panel) mRNA levels in mouse primary hepatocytes treated with BMP6 or metformin and infected with Ad-GFP, Ad-AMPK ca or Ad-Flag-SHP for 24 h +, 10 MOI. Data are presented as means ± SD. Arrows show locations of molecular weight markers. The experiment was repeated on a minimum of three separate occasions. Western blot images were cropped with a grey cropping line. All gels for Western blot analysis were run under the same experimental conditions. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed Student t-test.
